It is sometimes desired to detect a rare variant or multiple rare variants of a DNA sequence in an abundant variant of that sequence. Rare variants are often mutations of a normal gene sequence, which is sometimes referred to as a “wild-type” sequence. Mutations, including particularly rare mutations, are commonly found in cancer-related genes, mitochondrial genes at a low heteroplasmic frequency, and genes from a small subpopulation of bacteria or viruses. Mutant alleles may have only a single change in the DNA sequence (e.g., a point mutation), and frequently they exist in very low abundance in samples containing a corresponding very abundant sequence. Detection of those rare mutations associated with diseases plays an increasingly important role for disease diagnosis and prognosis in clinical practice. For example, point mutations in tuberculosis (TB) can generate drug resistance, making it difficult to select the right drug to use and prolonging treatment. Somatic mutations are useful biomarkers for the early detection of cancer or a prediction of the response or resistance to certain oncology drugs. Mutation in codons 12 and 13 of the KRAS gene occurs in 80-90% of pancreatic cancer and 35-50% of colorectal cancer. And mutations in the EGFR gene or the KRAS gene have been associated with the response or resistance to certain oncology drugs. Recently, the American Society of Clinical Oncology (ASCO) and National Comprehensive Cancer Network has updated its guidelines with a recommendation that therapies including panitumumab or cetuximab be limited to patients with wild-type KRAS for patients with advanced or metastatic colon or rectal cancer. Furthermore, due to the difficulty in accessing to the tumor region, there is an increased requirement to get the tumor cells from blood. Sensitive assays to detect rare mutations in the huge background of wild type allele are critically needed.
Numerous approaches have been developed in the attempt to detect somatic DNA mutations and minority alleles by real-time PCR (polymerase chain reaction) methods. These approaches, many of which are reviewed in Milbury, C. A. et al., PCR-Based Methods for Enrichment of Minority Alleles and mutations (2009) Clin. Chem. 55, pages 632-640, include allele-specific competitive blocker PCR, blocker-PCR, real-time genotyping with locked nucleic acids (LNA) or peptide nucleic acid (PNA), clamp PCR, and restriction enzymes in conjunction with real-time PCR, and allele-specific kinetic PCR in conjunction with modified polymerases. Additional methods include ARMS-PCR, TaqMAMA, and FLAG-PCR. Methods utilizing many of these approaches require either the use of modified bases, special enzymes, or additional proprietary reagents or procedures. In addition, most use a primer that is matched (that is, fully complementary) to a certain mutant sequence and mismatched to the corresponding wild-type sequence, the intent being to amplify only the mutant sequence such that the final amplification product would all be essentially only the mutant-type sequence. In methods that hybridize a primer to a mutation point, mismatches are erased during amplification. PNA or LNA clamp PCR do not rely on a mutant-specific primer, but the manufacture of PNA or LNA oligonucleotides is expensive compared to the manufacture of DNA oligonucleotides. Also, to determine whether a negative result means that a sample contains no mutant or whether there was a failure of amplification, a parallel sample without the PNA or LNA must be run. Existing approaches have proven to be less than satisfactory due to expense, high lower limit of detection, or both. A significant problem with existing selective-amplification assays is the occurrence of false positives caused by errors committed by Taq DNA polymerase. One way to decrease the error rate is to use a high fidelity polymerase such as Pfu or High Fidelity Taq. However, PCR amplification with such enzymes is less efficient, and the optimization of PCR conditions for those high-fidelity polymerases is quite difficult.
A PCR amplification method that preferentially amplifies mutant alleles but does not require an enzyme other than Taq DNA polymerase and provides an amplification product that can be sequenced is COLD-PCR (see Milbury et al., supra). COLD-PCR utilizes a precisely controlled PCR denaturation temperature, for example, 86.5° C., to preferentially denature duplexes, which may be heteroduplexes, containing mutant extension products and thereby preferentially amplify mutant sequences. Disadvantages of this method include a need for very precise temperature control, experimental fine tuning of the denaturation temperature, and variability from mutation to mutation (id). COLD-PCR also does not address the problem of false positives caused by errors committed by Taq DNA polymerase.